(1) The activation constant of cyclic nucleotide phosphodiesterase by calmodulin has been determined by several methods. No cooperativity was detected in the interaction of these two proteins. Two calmodulin binding sites per phosphodiesterase molecule are demonstrated by the Job plot. (2) Ca 2 plus binding to calmodulin has been carried out by following the intrinsic Tyr fluorescence of calmodulin or by using the fluorescence change of dansylated troponin C as an indicator for the free Ca 2 plus. Initial positive cooperativity followed by negative cooperativity in Ca 2 plus binding was revealed in these studies. (3) Kinetic studies showed that all four Ca2 plus sites are required for forming the activated phosphodiesterase-calmodulin complex. A 100,000-fold increase in the affinity between phosphodiesterase and calmodulin can be achieved by a 20-fold preferential binding of each of the four Ca 2 plus to be the protein-protein complex. The strong cooperativity of the four Ca 2 plus sites also permits the inactivation of phosphodiesterase to be turned on and off by a small change in Ca 2 plus concentration.